The use of radiometal-labeled small molecules conjugated to targeting proteins and peptides as diagnostic agents has been increasing. A major challenge in developing radiometal-based radiopharmaceuticals is ensuring metal chelate stability in vivo to avoid decreased contrast and increased radiation doses to non- target tissues and organs. Our current research has demonstrated the in vivo superiority of cross-bridged ligands (chelators) as carriers for copper radionuclides compared to traditional macrocyclic chelators, resulting in improved target uptake and pharmacokinetics of their chelator-peptide conjugates. To build on this advance, we propose to develop a second generation of cross-bridged chelators for copper-, indium- and gallium-based radiopharmaceuticals for diagnostic imaging and targeted radiotherapy of cancer. We will design pendant-armed cross-bridged cyclam and cyclen chelators that have convenient synthetic routes, enhanced radiometal binding ability, and more rapid complexation kinetics to enable efficient radiolabeling of proteins under mild conditions. We will prepare their metal complexes, assay their kinetic and thermodynamic properties, and verify correlations between these properties and in vivo behavior. In Aim (I), new cross-bridged chelators will be synthesized. In Aim (II), their copper, gallium, and indium complexes will be prepared and evaluated using acid inertness, electrochemical (Cu only) and stability constant criteria. Our working hypothesis, based on results from the current grant, is that ligands with a single anionic pendant arm are sufficient to ensure superior biological behavior, freeing the other arm for conjugation to targeting moieties. In Aim (III), the most promising ligands will be labeled with Cu-64 (Ga-67,68 and ln-111) and evaluated in vitro and in vivo for stability and biological clearance. Bifunctional chelators will be synthesized and conjugated to the somatostatin analog Tyr3-octreotate. Biological evaluation of these in tumor-bearing rats will test Aim (II) predictions to optimize ligand design. Development of a second-generation of cross- bridged tetraamine chelators for metal radionuclides will advance the viability of copper, gallium and indium radiopharmaceuticals for diagnostic imaging and therapy of cancer. These will have a wide array of applications in small-molecule-, peptide- and protein-based imaging and therapy agents. Relevance: Effective detection and therapy of cancer by metal-based radiopharmaceuticals depend critically on the intact delivery of these drugs to target organs. We aim to develop strong-binding radiometal carriers to ensure safe delivery of copper, gallium, and indium radioisotopes for both diagnosis and treatment of cancer.